The present invention relates to a newly identified polynucleotide as well as to the polypeptide encoded for by the polynucleotide, and to their production and uses, as well as their variants and their uses. In addition, the present invention relates to a method for producing melleolides and related fungal sesquiterpenoids aryl esters by using the newly identified polynucleotide/polypeptide.
Melleolides are characteristic secondary metabolites produced by the homobasidiomyceste genus Armillaria, the species of which are not only regarded as edible mushrooms, but many species are notorious forest parasites, reflecting their ability to form rhizomorphs that allow them to grow across nutrient-poor areas.
Melleolides are protoilludene type sesquiterpenoids and have a potent antimicrobial and cytotoxic activity. Up to now, there are approximately 50 known melleolides and they are almost exclusively produced by this fungal genus. Each molecule comprises a tricyclic sesquiterpenoid skeleton linked to an orsellinic acid-like polyketide side chain via an ester bond. The biosynthesis of protoilludene is thought to involve cyclization of the universal sesquiterpenoid precursor farnesyl diphosphate to protoilluden followed by further modification by cytochrome P450 monooxygenases and subsequent attachment of the polyketide side chain.
Whilst the biosynthesis of some terpenes, in particular of plants origin—like menthol, artemisinin or taxol—have been extensively studied in the past, little is known about the synthesis of the vast majority, in particular of terpenes produced by fungi.
The biosynthesis of all terpenes begins with the cyclization and rearrangement of one of three universal precursors, geranyl diphophate, farnesyl diphosphate, or geranylgeranyl diphosphate, to yield monoterpenoids, sesquiterpenoids or diterpenoids, respectively. These cyclization reactions, which are catalyzed by terpene synthases, are among the most complex chemical reactions known in nature.
Generally, plant and fungal terpene synthases show only low level of sequence identity, and whereas several terpene synthases have been isolated from plants, only a few have been reported from microbes. Also, only a very limited number of fungal sesquiterpenoid synthases have been cloned and functionally characterized, including, e.g., trichodiene synthase, aristolochene synthase, and presilphiperfolan-8b-ol synthase. Furthermore, six sesquiterpene synthases from Coprinopsis cinerea (Coprinus cinereus) yielding germacrene A, α-muurolene, δ-cadinene and α-cuprenene as major products have recently been characterized.
The biosynthesis of protoilludene type sesquiterpenoids—also known as melleolides, see above—is thought to involve cyclization of the universal sesquiterpenoid precursor farnesyl diphosphate to protoilluden followed by further modification by cytochrome P450 monooxygenases and subsequent attachment of the polyketide chain.
Since in particular melleolides, in medicine, are of high interest due to their antimicrobial and cytotoxic activity, it would be desirable to have them specifically produced or enriched in a controlled way.